Mass spectrometric imaging is a technique for investigating the distribution of a substance having a specific mass-to-charge ratio (m/z) by performing a mass analysis on each of a plurality of micro areas within a two-dimensional area on a sample, such as a piece of biological tissue. This technique is expected to be applied, for example, in drug discovery, biomarker discovery, and investigation on the causes of various diseases. Mass spectrometers designed for mass spectrometric imaging are generally referred to as imaging mass spectrometers. This device may also be called a mass microscope since its operation normally includes performing a microscopic observation of an arbitrary area on the sample, selecting a region of interest based on the observed image, and performing a mass analysis of the selected region. For example, the configurations of commonly known mass microscopes and analysis examples obtained with those mass microscopes are disclosed in Patent Document 1 as well as Non-Patent Documents 1 and 2.
A mass microscope is basically composed of a microscopic observation means for performing a microscopic observation of a two-dimensional area on a sample and a mass analysis means for performing a mass analysis for each of a plurality of portions within the two-dimensional area on the sample. The microscopic observation means can be divided into two major types: One type has an imaging means (e.g. a CCD camera) and a display unit (e.g. a monitor) with a screen on which an image taken with the imaging means can be displayed, thus allowing an operator to observe a sample image; the other type is a normal microscope having an eyepiece. The mass analysis means includes an ionization means for ionizing a component contained in a sample, an ion separation-detection means for separating the ions originating from the sample according to their mass-to-charge ratio and detecting each ion, and an ion transport means for guiding and transporting the ions generated from the sample to the ion separation-detection means.
The ionization means is typically a matrix assisted laser desorption ion source (MALDI ion source), a matrix-less laser desorption ion source (LDI), or a similar device. In these types of ion sources, a thin laser beam is thrown onto the sample, whereupon ions originating from sample components are generated at around the portion irradiated with the laser beam. The generated ions are extracted from the space near the sample by the action of an electric field and transferred to the ion separation-detection means via the ion transport means, such as an ion lens.
In the case where the ionization is performed under vacuum atmosphere, the electrodes and ion transport optical system for forming an electric field for extracting and accelerating ions generated from the sample are normally located above the sample placed on a sample plate. On the other hand, if the ionization is performed under atmospheric pressure, an ion intake port, which is used for drawing ions from the atmospheric pressure into a vacuum atmosphere where the ion separation-detection means is disposed, is arranged opposite to the sample. In any of these configurations, if an attempt is made to place the microscopic observation means above the sample to observe its surface, at least a portion of the aforementioned components of the mass analysis means spatially interferes with the microscopic observation means. Furthermore, such an arrangement may cause a decrease in the amount of ions supplied to the mass analysis due to the presence of the microscopic observation image in the path of the ions. To avoid such interference, various kinds of configurations have been proposed for the mass microscope.
For example, in the mass microscope shown in FIGS. 5-7 of Patent Document 1, an observation optical system is arranged so that a sample placed on a sample plate will be observed obliquely rather than from directly above (i.e. in the direction normal to the sample plate). This arrangement prevents the interference between the components of the microscopic observation means and those of the mass analysis means as well as the interference between the optical observation path and the transport path of the ions generated from the sample.
However, when the sample is observed from obliquely above rather than from directly above, the observed image becomes distorted, making it difficult to correctly perform the morphological observation of the sample. Furthermore, in some cases, the oblique observation allows only a limited portion of the visual field to come into focus, thus reducing the effective visual field. Another problem may result from the fact that the operating distance of the observation optical system inevitably becomes large to avoid the spatial interference between the observational optical path and the ion transport path or ion intake unit. Increasing the operating distance lowers the spatial resolution of the observed image, which may unfavorably affect the task of correctly selecting a desired area for the mass analysis.
In the mass microscope shown in FIG. 8 of Patent Document 1, a special type of observation optical system having an aperture for allowing the passage of ions is located directly above the sample. This optical system is designed so that a sample image can be laterally extracted for visual observation while allowing ions to be transported upwards through the ion-passing aperture and supplied for mass analysis. A mass microscope having such a configuration can create an image of the sample observed from directly above.
However, the presence of the ion-passing aperture at around the center of the observation optical system may cause a decrease in the contrast of the observed image at the center of the image or a defect in the visual field. Furthermore, the ions generated from the sample will not always travel in the direction normal to the sample plate; a portion of those ions will inevitably be spread away to some extent. This means that a portion of the ions generated from the sample do not pass through the ion-passing aperture but collide with the observation optical system, which may decrease the amount of ions supplied for the mass analysis and prevent the detection sensitivity from being sufficiently high. Another problem is that the laser irradiation causes various matters (e.g. fine particles), other than the ions, to be scattered from the sample and adhere to the observation optical system. Such contaminants may blur the observed image or cause a visual-field defect. Furthermore, the aforementioned special observation optical system having an uncommon construction may be considerably expensive.
In the mass microscopes shown in FIGS. 1 and 4 of Patent Document 1 or in Non-Patent Documents 1 and 2, the stage on which a sample is to be placed has a larger movable area so that the stage can be moved between the position for microscopic observation and the position for mass analysis. Since the observing position and the analyzing position are separated, it is possible to arrange the microscopic observation means above the observing position and the mass analysis means above the analyzing position so as to prevent the spatial interference between the components of these two means. Accordingly, a high-quality sample image observed from above can be obtained.
However, due to the separation between the observing position and the analyzing position, it is impossible to obtain a real-time image of the sample while the mass analysis of the sample is underway. Accordingly, the analysis operator cannot directly and visually check the irradiation point of the laser beam on the sample, which contributes to an uncertainty in the analyzing position. Furthermore, even if the sample is significantly consumed or damaged during the ionization process, the sample is separated from the sample plate, or an impurity (e.g. dust) sticks to the sample surface, the analysis operator cannot notice the problem during the analysis, continuing the analysis in vain. Furthermore, providing the stage with a large movable area will additionally increase the cost of the system.